Effects Of Intracellular Porphyromonas Gingivalis On Cell Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Cells

Background and objectivePeriodontal disease is the leading cause of tooth loss in adults. It is now well established that periodontal disease is a bacterially inflammatory disease induced by microorganisms in dental plaque. Microorganisms are essential in the destruction of periodontal tissues, among which Porphyromonas gingivalis (P. gingivalis) is widely considered as one of the most important pathogenic bacterium of periodontal disease.Previous reports demonstrated that P. gingivalis could adhere to cheek mucous membrane, periodontal pocket and other oral bacteria by using filamentous surface appendages or fimbriae. A panel of virulence factors produced by P. gingivalis included lipopolysaccharide (LPS), collagenase, gingipains etc. These are molecules that make destruction on periodontal tissues by reducing attachment of periodontal tissues and increasing absorption of allveolar bone, resulting in the loss of tooth finally. Besides, as part of its strategies for survival in the host, P. gingivalis was able to evade from the innate immune by invading cells and tissues. P. gingivalis in the host cells could survive for a long time, and spread to neighboring cells by intercellular bridge. Yet so far the etiology and mechanism of periodontal disease induced by P. gingivalis were still unclear. By examining some kinds of virulence factors, previous studies have tried to explore the mechanism of periodontal disease to some extent. However, researchs simply focusing single component always failed to the unveil the truth. For example, LPS produced by microorganisms within the cells tended to take an effect until lysis occurred.Therefore, in this study we aim to determine the ability of P. gingivalis to invade hPDLCs, and the effect of P. gingivalis on biologic structure, cells proliferation and osteogenic differentiation in vitro, which may help us explore the further pathogenesis of periodontal disease.Chapter 1 Primary culture and identification of human periodontal ligament cells in vitroObjective:To isolate and culture human periodontal ligament cells (hPDLCs) in vitro, and identify their morphological, histological and growth characteristics.Methods:1. Healthy premolars were extracted in the condition of asepsis from orthodontic patients between 12 to 25 years old. The primary cells were isolated from periodontal tissues and cultivated by modified collagenase tissue-explant method, then passaged when the cells completely covered the bottom of culture flasks. Morphological and growth characteristics of the third generation of hPDLCs were observed by inverted phase contrast microscope.2. Vimentin and ceratin of the third passage of hPDLCs were stained by immunohistochemical SP method to determine the source of hPDLCs.3. Cell viability of the third passage of hPDLCs was detected by MTT assay for 7 days, and growth curve was made afterward.Results:1. After 5 to 10 days of primary cultures, fibroblasts were observed migrating from the tissue explants. Microscopy showed that the cultured cells were fibroblast-like appearance with a stellate or long spindle shape with full body, uniform cytoplasm and round or oval nucleus containing visible nucleoli. Within 24 hours of cultures, the passaged cells tended to adhere to the culture plate surface, and grew at a faster rate with transforming their appearance into irregular round, polygon and spindle shape.2. By immunohistochemical analysis, the cultured cells were positive to antibodies against vimentin, and negative to antibodies against ceratin, indicating themselves to be mesoderm-derived fibroblasts.3. After 1 to 2 days of primary culture, the growth rate of these cells was slow, and accelerated after 3 days, then reached the stationary phase about 7 days later, showing a grow pattern of primary cell culture in vitro with growth curve of inverted S appearance, which also indicated that the proliferation viability of these cells of is satisfactoryConclusion:The modified collagenase tissue-explant method is an ideal method to obtain and culture periodontal ligament cells having typical characteristics.Chapter 2 Culture and identification of porphysomanas gingivalis in vitroObjective:To culture P. gingivalis in vitro,and identify them by morphological observation and 16s rDNA sequencing.Methods:P. gingivalis ATCC33277 were inoculated into broth-enriched brain heart infusion (BHI) with sheep blood agar medium supplemented with 50mL/L defibrinated horse blood,5mg/L paraxin and lmg/L vitamin K3 under anaerobic conditions (10% CO2,10% H2,80% N2) at 37℃. Structural and growth characteristics of P. gingivalis were observed by microscopy, and Gram’s staining was performed. After incubated for 7 days, single colony of P. gingivalis was picked into BHI liquid medium, and was cultured under anaerobic conditions at 37℃ for 24 hours. 1mL sample of P. gingivalis mixture was identified by 16s rDNA sequencing.Results:1. After cultured for 3 days, P. gingivalis were observed as a white, hard, 0.5～1mm in diameter colony which was protuberating and adhering on the surface of the medium.7 days later, the color changed into black. Gram’s stain proved that P. gingivalis was a red Gram-negative coccobacillus.2. The sequencing result proved that P. gingivalis ATCC33277 had been chosen.Conclusion:P. gingivalis were acquired successfully by anaerobic method and could be applied to further study.Chapter 3 Comparative study on the ability of porphysomanas gingivalis to invade human periodontal ligament cellsObjective:To compare the ability of P. gingivalis to invade hPDLCs at different multiplicity of infection (MOI) and invasive time and establish a model of intracellular infection of hPDLCs by live P. gingivalis in vitro for further study.Methods:1. hPDLCs were co-cultured with P. gingivalis at MOI 10 and 100 for 90min,8h, and 24h separately. Intracellular invasion assay and flow cytometry were employed to compare the ability of P. gingivalis to invade hPDLCs.2. After hPDLCs were co-cultured with P. gingivalis labeled by SYTO-9 for 24 hours, F-actin from cytoskeleton of hPDLCs were stained by Alexa Fluor(?)594 Phalloidin and nuclear was stained by DAPI. Then intracellular invasion of P. gingivalis was observed by laser scanning confocal microscope (LSCM).Results:1. When P. gingivalis were co-cultured with hPDLCs for 90min, they were found in the cell. Invasive ability reached at maximum after 24h, while showing no difference between MOI 10 and 100.2. P. gingivalis labeled by SYTO-9 emitted green fluorescence under LSFM. They located in cytoplasm, surrounding with nuclear mostly. Conclusions:A model of hPDLCs invaded by live P. gingivalis was successfully established in vitro. The best invasive time was 24 hours. However, invasive ability showed no difference between MOI 10 and 100.Chapter 4 Effects of Porphyromonas gingivalis on cell proliferation, osteogenic differentiation of human periodontal ligament cellsObjective:To explore the effects of intracellular P. gingivalis biologic structure, cell proliferation, osteogenic differentiation of hPDLCs.Methods:1. The effect of P. gingivalis on biologic structure of human periodontal ligament cells was observed by transmission electron microscope (TEM).2. The cell proliferation ability of hPDLCs was detected by flow cytometry after labeled by CFDA-SE and then co-cultured with P. gingivalis for 8,24,48,72 hours respectively.3. The changes of apoptosis hPDLCs were detected by flow cytometry (FCM) after invaded by P. gingivalis.4. After co-culture with P. gingivalis labeled by SYTO-9 for 24 hours, the infected hPDLCs were sorted out by fluorescence-activated cell sorting (FACS). After induced by osteogenic differentiation medium, alizarin red S staining was used for detecting mineralization nodules deposition.5. qRT-PCR and western blot were employed to detect the expression level of Runx2 mRNA and protein respectively.Results:1. TEM showed that P. gingivalis could survive afer invading into hPDLCs, and none of them have changed the integrity of hPDLCs.2. FCM showed that hPDLCs infected by P. gingivalis had no alteration on cell proliferation at different time, except for a few self-renewal.3. FCM analysis showed that apoptosis detected by Annexin V-FITC/PI kit was not induced after hPDLCs invaded by P. gingivalis.4. After induced by osteogenic differentiation medium, hPDLCs invaded by P. gingivalis showed an obvious decrease in mineralization nodules deposition.5. qRT-PCR and western blot indicated that Runx2 of hPDLCs invaded by P. gingivalis was down-regulated on both mRNA and protein level significantly.Conclusion:P. gingivalis cannot affect the biologic structure, cell apoptosis as well as proliferation of hPDLCs, but inhibits their osteogenic differentiation by probable down-regulation of Runx2.